Colorectal cancer (CRC) is the third leading cause of cancer-related death in the U.S. Although advances in CRC screening, surgical therapies and medical management have improved clinical outcomes, there still is a major need to develop better medical therapies for CRC, particularly among those with advanced local or metastatic disease. The 5-year survival rate of victims with stage IV disease is a dismal 5%. There is increasing evidence that most solid tumors, including CRC, have a subpopulation of tumor-initiating cells termed tumor stem cells (TSCs). Epithelial-mesenchymal transition (EMT) is a key feature in cancer invasion and metastasis and is linked to a TSC phenotype in CRC. The COARE team has demonstrated that the tumor stem cell marker DCLK1 is significantly upregulated in CRC and is a central regulator of key oncogenic pathways and EMT. Recent studies have definitively confirmed DCLK1's TSC status in the Apcmin/+ model of intestinal neoplasia. COARE's pre-clinical experimental data shows that therapeutic targeting of cells that overexpress DCLK1 arrests tumor growth in xenografts and reduces the size and number of adenomas in Apcmin/+ mice. DCLK1 signaling inhibition triggers the induction/activation of several critical endogenous tumor-suppressor pathways within the tumor, which in turn regulate oncogenic pathways and processes and EMT-related transcription factors. DCLK1 is a unique TSC marker because it contains an extracellular C-terminal domain. Antibody-drug conjugates (ADCs) allow the specific targeting of tumor cell-surface expressed antigens with cytotoxins, with the potential benefits of enhanced efficacy and reduced off-target toxicity. COARE's CBT-3112 is a Paclitaxel-based ADC targeting the DCLK1 TSC/CTC antigen in CRC with the potential to improve outcomes in locally advanced/metastatic CRC. We will pursue three Specific Aims. Aim 1: Demonstrate that CBT-3112 will effectively deliver a cytotoxic payload to CRC cells resulting in cell death. Aim 2: Demonstrate that CBT-3112 will suppress tumor growth in TSC-derived tumor isografts. Aim 3: Demonstrate that CBT-3112 will suppress tumor growth in xenograft models of CRC with and without 5-fluorouracil and prevent in vivo metastasis. Test of Feasibility: CBT-3112 should induce cell death and inhibit proliferation comparable to Paclitaxel (PTX) alone. Additionally, it should significantly inhibit cell invasiveness and demonstrate internalization of PTX. In TSC isografts, CBT-3112 treatment should precipitate a significant reduction (>50%) in tumor growth and >40% induction of tumor suppressors and loss of downstream oncogenic signaling compared to controls. In the tumor xenografts, CBT- 3112 should significantly reduce tumor volume alone, sensitize tumors to 5-fluorouracil (5-FU), and prevent engraftment of metastatic CRC cells in an in vivo metastasis assay. Phase I success will lead to a Phase II project focused on humanizing DCLK1 mAb, completing detailed pharmacokinetics and in vivo toxicity analyses, and preparing for IND/clinical trial work to set th stage for ultimate commercialization.